April 20, 2016

Rhizosphere soil environment

Philippot, L. et.al. (2013). Nat Rev Micro

Change of bacterial diversity in plant compartments

Nodule metagenome (Brazil)

Márcio Leite

Sequencing details

Feature Nodule (BR) Endo (NL)
Platform MiSeq MiSeq + HiSeq
Read length 300 300
Samples 18 10
Raw 8.32 Gbp 191.6 Gbp
QC 4.16 Gbp 47.2 Gbp
Gbp sample 0.23 4.72

Glueing tools together into a re-usable workflow

Assembly statistics

Feature Nodule Endo
assembler megahit SPAdes
# contigs 74,507 1,627,767
% mapped 99.65 98.73
properly paired 78.16 88.20
N50 10,631 1,652

Sangwan, N. et.al. (2016). Microbiome

Mads Albertsen

Coverage - GC plots for the nodule metagenome

Contigs have also different tetranucleotide frequencies

Details of the most important bins

Bin Id Marker Lineage Completeness Contamination Length (Mbp) # Contigs
bin.3 f__Paenibacillaceae 99.85 0.02 6.4 71
bin.8 o__Actinomycetales 99.81 0 3.3 57
bin.7 f__Moraxellaceae 99.51 0.82 2.9 61
bin.9 f__Moraxellaceae 99.45 0.14 2.9 72
bin.4 g__Burkholderia 98.46 0.57 7.4 140
bin.10 o__Bacillales 97.80 1.34 4.9 139

Classification by essential and 16S rRNA genes

Issues with 16S rRNA

  • Many 16S rRNA sequences are only partial assembled into short contigs
  • The sequence characteristics of 16S sequences are different compared to protein coding genes:
    • average coverage and sequence composition
    • more conserved
  • Therefore they are missed by automated binning methods
  • Assembly specific for 16 rRNA genes with tools like EMIRGE or Reago would help the annotation genome bins (future work)

The endosphere is a more complex environment

Conclusions

  • Plant root-associated compartments have a different diversity
  • We are able to extract draft genomes from plant root and nodule metagenomes
  • Visualization of contig characteristics enables
    • manual binning for low-diversity samples (plant nodules)
    • verification of automated binning methods
  • Creating genome bins together with its rRNA sequence remains problematic when using only coverage and sequence composition

Acknowledgements

  • Vittorio Tracanna (Poster#77)
  • Noriko Cassman (Poster#70)
  • Victor Carrion
  • Marcio Leite
  • Juan Perez
  • Victor de Jager
  • Eiko Kuramae
  • Jos Raaijmakers

Endophytic metagenome (NL)

Weller, D.M. et.al (2002). Annual Review of Phytopathology

pictures victorc

Victor Carrión